The present invention relates to the field of processes for producing food and biotechnological products by implementing one or more fermentation steps.
The methods envisioned are very varied and can be divided up into two major groups:                methods for making fermented food products such as fermented milk products (yoghurts, fromage blanc, etc.), fermented drinks (beers, wine, etc.),        methods for making a biomass (lactic ferments, yeasts, etc.) and metabolites (food ingredients, molecules of interest such as enzymes, amino acids, medicaments, ethanol, etc.).        
The invention endeavors to propose novel operating conditions that make it possible, depending on the case in question, to improve in particular the properties of the products thus produced, and in particular their microbiological properties, sensory properties, physicochemical properties, etc., or else the yields, purities, etc.
As will be seen below in greater detail, the present invention proposes a novel method for carrying out such processes by controlling and regulating the redox potential of the medium under consideration at a given redox potential level (redox level defined in each case so as to have an optimum desired effect), at one or more key points of the method, by controlled additions of one or more suitable gases or gas mixtures.
Redox potential measurements can be obtained in a medium using any available means, such as, for example, redox probes which make it possible to carry out direct and continuous measurements in liquid or semi-liquid media, or else to carry them out indirectly through measurements of dissolved gas content, for example of dissolved hydrogen in the medium.
The monitoring and the regulating of the redox potential at certain key steps of the process make it possible, for example, to carry out steps or phases of the process under stable reducing conditions or to alternate, during the process, between steps under reducing conditions and steps under oxidizing conditions.
A condition is considered to be oxidizing or reducing relative to the redox potential of the medium before the adjustment with the gases. Thus, a condition will be termed reducing when the oxidoreduction potential is less than the initial value before its adjustment and its regulation (irrespective of whether or not the potential reached is negative). Conversely, a condition will be termed oxidizing when the oxidoreduction potential is greater than the initial value before its adjustment and its regulation (irrespective of whether or not the potential reached is positive).
It will be recalled that oxidoreductions are essential steps in cell anabolism and catabolism reactions, for which the direction of the exchanges is determined by the oxidoreduction potential (hereinafter Eh). The Eh is a fermentation state parameter; variation thereof modifies the physicochemical environment of microorganisms. The metabolic activities and the physiology of microorganisms are determined by the intracellular pH (pHin) which will condition the activity of the enzymes and the accessibility of certain substrates and cofactors in the metabolic reactions. The pHin depends on the extracellular pH (pHex) and on the ability of the microorganism to maintain a certain cellular homeostasis. The difference between the pHin and the pHex will also modify the value of the proton motive force ΔμH+, which is in particular involved in the exchanges of the microbial cell with the exterior. The Eh and pHin parameters are intimately linked; thus, the energy found in high-potential compounds, such as adenosine triphosphate (ATP), and gained by substrate catabolism may be used by the cell in order to maintain its pHin (and therefore its ΔpH) by virtue of membrane ATPases.
According to Urbach et al., in 1995 (“Contribution of lactic acid bacteria to flavour compound formation in dairy products”, International Dairy Journal, 5: 877-903), lactic acid bacteria are widely involved in the production of the flavor compounds of fermented dairy products; they convert lactose to lactic acid; this results in the production of diacetyl and of acetaldehyde, which are the principle flavor compounds of fermented milks and of fromage frais. The Eh is an environmental parameter which will be able to condition the metabolic activities of microorganisms and in particular their ability to synthesize flavor molecules. In particular, it has been shown, for emmental and cheddar, that good-quality cheeses have a low oxidoreduction potential.
The Eh is a physicochemical parameter which, by virtue of its nature, acts on all media, provided that the latter contain at least one molecule which can pass from an oxidized state to a reduced state and vice versa. For this reason, its effect can be seen on all cellular functions. Its action has been shown on various types of bacterial strains; by way of illustration:                the addition of chemical reducing agents to culture media has made it possible to significantly modify growth and metabolic fluxes in Corynebacterium glutamicum, Clostridium acetobutylicum, Sporidiobolus ruinenii and Escherichia coli;         a reducing Eh fixed by gases has made it possible to modify the metabolic fluxes in Saccharomyces cerevisiae with an increase in the glycerol/ethanol ratio and the accumulation of storage sugars with an increase in yeast survival during conservation.        
In the industrial environment, the Eh is already indirectly taken into account through oxygen, the inhibitory effect of which on lactic acid bacteria has been well identified. This effect is due to their inability to synthesize cytochromes and enzymes containing a heme nucleus.
It is, moreover, known that it is also possible, by acting on the Eh, to modify the survival of probiotic ferments, metabolic fluxes, and the production and/or the stability of flavor molecules. All these results were obtained following modification of the Eh by the microorganisms themselves, by oxidoreductive molecules, or by thermal treatment.
In the field of the use of gas mixtures in lactic acid bacteria fermentation media, mention may also be made of the studies by Henriksen et al., published in Letters in Applied Microbiology in 2000 (Vol. 30 p. 415-418), which focus on the growth of lactic acid bacteria, and showed that, when the cultures were swept with nitrogen, growth was greatly slowed, whereas the addition of minute amounts of CO2 in this case caused the growth to begin again in exponential form.
The present invention therefore relates to a process for producing a food or biotechnological product, implementing one or more steps, wherein one or more of the steps uses a medium, one or more of the steps that uses a medium being a fermentation step, said process consisting, during at least one of the steps thereof, in controlling the redox potential of the medium of the step in question, and being characterized in that it is conducted in the following way:                regulating at a predetermined setpoint level the redox potential of the medium of the step of which the redox potential is controlled with controlled additions of a process gas to the medium in question, and        proceeding to the step that follows said step in question in the process when said setpoint value is reached,so as to carry out at least one of said steps of the process under reducing conditions and at least one of said steps of the process under oxidizing conditions.        
In the subsequent text, reference will be made without distinction to steps or phases constituting the process, or alternatively to phases constituting a step of the process.
The process according to the invention may, moreover, adopt one or more of the following technical characteristics:                the process is a process for producing a fermented milk product, and the regulating of the redox potential is carried out in several of the steps so as to sequence the oxidation and reduction phases in the following way:                    the redox potential is regulated so as to set up reducing conditions at one or more points of the process located upstream of the pasteurization phase;            the redox potential is regulated so as to set up oxidizing conditions at one or more points of the process located downstream of the pasteurization;                        the process is a process for producing a fermented milk product, and the regulating of the redox potential is carried out in several of the steps so as to sequence the oxidation and reduction phases in the following way:                    the redox potential is regulated so as to set up oxidizing conditions at one or more points of the process located upstream of the pasteurization phase;            the redox potential is regulated so as to set up reducing conditions at one or more points of the process located downstream of the pasteurization;                        the fermented milk product is a yoghurt;        said conducting of the process is done in such a way that, for at least one of said fermentation steps, controlled addition of a process gas make it possible to alternate between phases of the fermentation in question under reducing conditions and phases of the fermentation in question under oxidizing conditions;        the process is a process for producing beer, and said conducting of the process allows the regulating of the redox potential during the fermentation to take place in two steps: in a first step, the fermentation takes place under regulated oxidizing conditions and in the presence of oxygen, so as to promote the growth of the yeast and a good physiological condition thereof, and in a second step, the redox potential is reduced to an optimal value so as to make it possible to improve the fermentation parameters, and also the sensory criteria;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and said conducting of the process makes it possible to regulate the redox potential of the medium at various successive values according to the various fermentation phases, so as to carry out a first phase under oxidizing conditions in order to promote the growth of the microbial strain through oxidizing conditions and, after a maximum biomass content has been obtained, to switch the fermenter to reducing conditions in order to initiate or intensify the production of one or more desired metabolites;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and said conducting of the process makes it possible to regulate the redox potential of the medium at various successive values according to the various fermentation phases, so as to carry out a first phase rendered slightly reducing, which is favorable to the growth of certain microorganisms and therefore to the production of a large biomass, followed by a more reducing phase which will make it possible to promote the production of desired flavor compounds;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and said conducting of the process makes it possible to modify and regulate the redox potential of the medium at a different value at the end of fermentation in order to adapt the metabolism or the physiology of the microorganisms so as to prepare them for a subsequent step;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and said conducting of the process makes it possible to change the redox potential level after the production of a desired metabolite so as to promote the excretion of the metabolite in question into the recovery medium;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and said conducting of the process makes it possible to change the redox potential level after the production of a precursor of a molecule of interest so as to promote a chemical reaction for obtaining the desired molecule of interest;        the process is a fermentation process in a fermenter, aimed at the production of biomass and/or of metabolites, and in that, subsequent to the fermentation, the following are carried out:        centrifugation and/or filtration and/or ultrafiltration steps aimed at recovering the biomass produced, or        purification steps on the fermentation medium in order to separate and concentrate metabolites,and said conducting of the process makes it possible to change the redox potential level after the separation of the medium containing the molecule of interest from the biomass (microbial cells) so as to promote the selective separation of the molecule of interest from the other compounds of the medium by promoting, for example, the binding to a resin either in the reduced form or in the oxidized form of the molecule of interest and elution thereof with a suitable solution.        
It is therefore proposed to control the redox potential at key places in the process using any available means such as, for example, redox probes. This control makes it possible to adjust the redox potential and to control the addition of gas to the medium, while at the same time accurately determining the moment when it is possible to stop the redox modification process.
It is possible to envision a system for automatically regulating the redox potential of the medium, since it is known, for example, that certain microorganisms, by virtue of their activity, modify the redox potential of a medium. It is thus possible to keep a stable redox potential for a given period of a fermentation.
It may also be possible, according to the process in question, to alternate between phases of reducing conditions and oxidizing conditions so as to promote, at defined moments in the process, the setting up of biochemical and/or biological reactions which, by virtue of the sequencing, make it possible to obtain a product with defined characteristics. Thus, by way of illustration, it may be advantageous to conduct a fermentation with a first phase rendered slightly reducing by the addition of nitrogen (driving off part of the dissolved oxygen) which is favorable to the growth of certain microorganisms and therefore to the production of a large biomass, followed by a more reducing phase (for example using a mixture containing hydrogen) which will make it possible, for example, to promote the production of desired flavor compounds such as, for example, the acetaldehyde produced by lactic acid bacteria.
As will have been understood from reading the above, the process gas would have to be chosen according to the process in question, the step in question, and the redox conditions that it is desired to reach, and it will therefore be possible to envision using a neutral gas such as nitrogen, argon, helium or carbon dioxide, and also an oxidizing gas such as oxygen or air, or alternatively a reducing gas such as hydrogen, or even a mixture of such gases.
It is accordingly possible to envision carrying out a regulation of the redox potential at the following points of the chain:    i) in the starting milk before fat correction,    j) in the milk mixture resulting from the addition of the additional ingredients to the milk;    k) before or after the step of homogenizing said milk mixture;    l) before or after the step of pasteurizing the milk mixture;    m) before or after the step of seeding the milk mixture with one or more strains of lactic acid bacteria;    n) before the packaging of the product in its final packaging.
In accordance with the invention, in this case of the production of yoghurts or fermented milk products, it is possible to envision sequencing the oxidation and reduction phases. In fact, without being at any time bound by the explanation which follows, it may be considered that the reducing values will be able to favorably adjust the conformation of the proteins, in particular the serum proteins, rich in sulfur molecules. The redox potential makes it possible to adjust the state of these molecules which are in the form of thiol groups or in the form of disulfide bridges, and play an essential role in the formation of the protein network after denaturation of the proteins during the pasteurization step. The use of reducing conditions upstream of the pasteurization phase will therefore make it possible to couple this effect with the effect of the heat on the proteins, resulting in the production of protein gels having a stable structure that will be favorable in particular to a limitation of the syneresis phenomena in yoghurts. Downstream of the pasteurization, it will then be advantageous to re-establish less reducing redox values (or redox values equal to the normal values of milk) which will allow the seeded bacteria to develop normally without influencing their metabolism and therefore without any organoleptic consequence.
It may also be advantageous to adjust a target value of the redox potential during the fermentation in order to intentionally influence the metabolism of the lactic ferments, and thus, for example, to direct the production of flavors toward the desired compounds.
By switching so as to finish with the redox potential of the finished product at a more reducing value than that of the fermentation, it will be possible to microbiologically stabilize the yoghurt and thus to more effectively preserve it against the possible development of certain yeasts or molds.
In the above text, examples of the production of yoghurts have more particularly been developed, but mention may also be made of the case of the production of fermented drinks such as beer, and also the case of the production of products in fermenters. This will be done below.
The case of beer production will therefore now be discussed.
Shown below will be the fact that, in the case of beer production, it is advantageous to conduct the process in such a way that the regulating of the redox potential during the fermentation takes place in two steps, in two phases. In a first step, the fermentation advantageously takes place under regulated oxidizing conditions and in the presence of oxygen, so as to promote the growth of the yeast and a good physiological condition thereof. In a second step, the redox potential is reduced to an optimal value so as to make it possible to improve the fermentation parameters and also the sensory criteria (flavors, foam retention).
It should be recalled that the brewing process typically comprises two fermentation steps:                the main fermentation: after aeration of the wort, the latter is seeded with a yeast of the Saccharomyces genus, which will, through fermentation, convert the fermentable sugars to alcohol and to carbon dioxide.        the secondary fermentation or “standing”: this step is carried out subsequent to the previous step by reducing the temperature of the medium to a temperature close to 0° C. for a period which varies from a few days to a few weeks. The young beer will become saturated with carbon dioxide, which will greatly contribute to its foaming character. It is also during this maturing phase that the beer clarifies and that its flavor matures.        
The examples which follow will show that it is advantageous to ferment the wort under oxidizing conditions at selected moments and under reducing conditions at selected moments.